Human replication licensing factor Cdt1 directly links mitotic kinetochores to spindle microtubules

Robust kinetochore-microtubule (kMT) attachment is critical for accurate chromosome segregation. G2/M-specific depletion of human Cdt1, a protein that localizes to kinetochores in an Ndc80 complex-dependent manner, leads to abnormal kMT attachments and mitotic arrest indicating an independent mitotic role for Cdt1 in addition to its prototypic DNA replication origin licensing function. Here, we show that Cdt1 directly binds to microtubules (MTs). Transiently expressed Cdt1 localizes to mitotic spindle MTs. Deletion mapping revealed that the C-terminus of Cdt1 carrying a winged-turn-helix domain is necessary but not sufficient for MT-binding. Mitotic kinase Aurora B interacts with and phosphorylates Cdt1. Aurora B-phosphomimetic Cdt1 exhibited attenuated MT-binding and its cellular expression induced defective kMT attachments and mitotic progression. Thus we provide evidence for a Cdt1-mediated MT-attachment site at kinetochores. We propose that Cdt1 links the loop domain of Ndc80 to MTs and enables an optimal MT-binding state for the complex.